La familia como titular del derecho al bienestar económico y a la

Figure 1. NEPC has a distinctive heterochromatin gene signature. (A-F) GSEA show the 675

enrichment of heterochromatin-associated genes in NEPC from (A) PDX models, (B) the 676

Beltran et al. 2011 cohort (23), (C) the Beltran et al. 2016 cohort (10), and in NE-like GEM

677

models derived from (D) Rb/Tp53 DKO or Pten/Rb/Tp53 TKO (11), (E) NPp53 CRPC with 678

NE differentiation (17), (F) and Nmyc overexpression (8). “NES” stands for normalized 679

enrichment score; FDR q values were calculated using 1000 gene permutations except for 680

(C), where Gsea preranked was applied. (G) Heatmap showing the hierarchical clustering 681

among all PDX samples suggests a unique upregulation of heterochromatin signature genes 682

in NEPC PDX tumors. (H) Weighted NE scores and heterochromatin scores of 683

adenocarcinoma and NEPC PDX tumors and two clinical cohorts. NE scores were 684

calculated based on the gene panels from Beltran et al. 2016 (10) and Lee et al. 2016 (18). 685

Heterochromatin scores were calculated based on the weighted gene panel from (G). 686

Scatter plots show the calculated score of each tumor sample, with lines indicating the mean 687

value and 95% CI. The p-values were calculated using the unpaired two-tail Student’s t-test. 688

See also Fig. S1. 689

Figure 2. HP1α expression is upregulated during NE transdifferentiation and in NEPC 690

PDX models. (A) A heatmap showing the gene expression changes in the LTL331/331R NE 691

transdifferentiation PDX model. Heterochromatin signature genes, AR signaling targets, and 692

NE markers are included. RNA-seq data from two individual samples at each time point was 693

used (pre: LTL331 pre-castration; Cx: 8 weeks post-castration; Rep: LTL331R NEPC 694

relapse). Average differences in expression between castrated and pre-castrated tumors (C 695

vs. P) are shown in a separate heatmap. (B-D) HP1α expression in PDX models, as 696

determined by (B) qRT-PCR, (C) IHC, and (D) Western blotting. Scatter plots show relative 697

mRNA expression or IHC score for each sample, with lines indicating median values. (E-G) 698

HP1α expression in the LTL331/331R castration-induced NE transdifferentiation PDX model, 699

as determined by (E) qRT-PCR, (F) Western blotting, and (G) IHC. Data show mean ± SEM 700

from three replicates. The p-values (B, C, E) were calculated with unpaired two-tail Student’s 701

t-tests. See also Fig. S2.

702

Figure 3. HP1α expression is upregulated in clinical NEPC samples and correlates 703

with poor prognosis in adenocarcinomas. (A-C) HP1α mRNA expression in NEPC vs. 704

adenocarcinoma from (A) the Beltran, et al. 2011 cohort (23), (B) the Beltran, et al. 2016 705

cohort (10), and (C) the Grasso, et al. 2012 cohort (24). Scatter plots show RNA expression 706

data of each sample, with lines indicating median values. The p-values were calculated 707

using unpaired two-tail Student’s t-test. (D) Staining intensity for the HP1α protein in primary 708

adenocarcinomas (n=103), CRPC-adenocarcinomas (n=120), and NEPC (n=8) as 709

determined by IHC of the VPC TMA. Box plots show the mean with whiskers representing 5- 710

95% percentile values. The p-values were calculated using unpaired two-tail Student’s t-test. 711

(E) Representative IHC images for the various staining intensities (0 to 3) are shown, with 712

the lower panels being magnifications of the selected regions in the upper panels. Scale 713

bars in the upper and lower panels represent 100 μm and 10 μm respectively. (F-G) Kaplan- 714

Meier survival analyses of estimated (F) relapse-free survival time based on the HP1α IHC 715

and (G) prostate-cancer specific survival time after first hormonal therapy based on HP1α 717

mRNA from metastatic adenocarcinomas in the Grasso, et al. 2012 cohort (n=33) (24). The 718

p-values were calculated using the log-rank test to determine the difference in outcomes

719

between patients with high (red) and low (black) HP1α expression. 720

Figure 4. HP1α is essential for the aggressive growth of NEPC cells and tumors. (A) 721

Stable knockdown of HP1α in NCI-H660 cells by lentiviral transduction. Changes to HP1α 722

mRNA and protein levels were determined by qRT-PCR and Western blotting respectively. 723

Bar graph shows mean ± SEM. The p-value was calculated by unpaired two-tail Student’s t- 724

test. (B) Cell growth assay of HP1α knockdown H660 cells as determined by crystal violet 725

staining. Stable cells were plated in four replicate wells for each time point and cell numbers 726

were determined based on the absorbance at O.D. 572 nm of crystal violet dissolved in 2% 727

SDS. Data is graphed as mean ± SEM. Representative images demonstrating cell numbers 728

at the final time point are shown. The p-value was calculated by two-way ANOVA. (C) 729

Colony formation assay of HP1α knockdown H660 cells as determined by crystal violet 730

staining. Cells were plated in four replicate wells for each stable line and colony numbers 731

were counted manually. Bar graph shows mean ± SEM. Representative images 732

demonstrating colony numbers are shown. The p-value was calculated by unpaired two-tail 733

Student’s t-test. (D) An EdU incorporation assay was performed by incubating stable HP1α 734

knockdown H660 cells with 10 μM EdU for 4 hours. EdU-labeled cells (red) and total cells 735

counterstained with DAPI (blue) were counted for at least 10 fields. Bar graph shows the 736

mean (EdU-positive ratio) ± SEM. Representative images are shown with the scale bar 737

representing 100μm. The p-values were calculated by unpaired two-tail Student’s t-test. (E) 738

An apoptosis assay measuring caspase-3 activity using the ApoLive-GloTM Multiplex 739

Reagent. Relative apoptosis was determined by the ratio of luminescence (caspase-3 740

activity) to fluorescence (AFC signal for viable cells). Bar graph shows mean ± SEM 741

(normalized to shC). The p-values were calculated by unpaired two-tail Student’s t-test. (F) 742

Cell death following stable HP1α knockdown in H660 cells was determined by flow 743

cytometry with 7-AAD staining. 10,000 single cells were collected for analysis with dead cells 744

being 7-AAD positive. Bar graph shows mean ± SEM (normalized to shC). The p-values 745

were calculated by unpaired two-tail Student’s t-test. (G) Apoptosis markers cleaved- 746

caspase 3 and cleaved-PARP1 as determined by Western blotting following stable HP1α 747

knockdown in H660 cells. Intact caspase 3 and PARP1 serve as controls. Cl stands for 748

“cleaved”. (H) Selected gene sets enriched in HP1α- versus control- knockdown H660 cells 749

as analyzed by GSEA. The x-axis represents normalized enrichment score (NES). FDR of all 750

gene sets is less than 0.05 calculated by 1000 permutations. (I-J) Stable H660 cell lines shC 751

and KD2 were each grafted into four NSG mice (eight tumors total) to assess in vivo 752

xenograft tumor growth. (I) Tumor volume was measured starting from when palpable tumor 753

appears to when mice were euthanized. Line graph shows mean ± SEM, with p-value 754

calculated by two-way ANOVA. Tumor images are shown in the right panel. (J) Fresh tumors 755

were also weighed at sample collection. Bar graph shows mean ± SEM with The p-value 756

was calculated by unpaired two-tail Student’s t-test. See also Fig. S3. 757

Figure 5. HP1α promotes NE transdifferentiation of prostatic adenocarcinoma cells 758

following ADT. (A) Ectopic expression of HP1α in LNCaP-V16D cells as determined by 759

overexpressing HP1α. Relative mRNA and protein expression of terminal NE markers (SYP, 761

CHGA, NSE, NCAM1) were detected by (B) qRT-PCR and (C) Western blotting in stable 762

cells cultured in complete medium (FBS), CSS, and CSS with 10 μM EnZ 14 days. Bar 763

graphs show mean ± SEM. The p-values were calculated by unpaired two-tail Student’s t- 764

tests comparing HP1α- to control- overexpressing cells. Relative band intensities as 765

determined by ImageJ are indicated, with actin serving as internal control. (D) A heatmap 766

comparing expression of NEPC marker genes in the indicated V16D cells upon EnZ 767

treatment. The select NEPC marker genes upregulated by HP1α overexpression are 768

similarly upregulated in clinical NEPCs in the Beltran, et al. 2011 cohort (23). (E) Top 10 769

pathways significantly enriched in HP1α overexpressing V16D cells compared to control 770

cells as analyzed with IPA. (F-G) Pearson correlation analysis of HP1α mRNA expression 771

and the expression of various NE markers in advanced PCa samples. (F) Correlation with 772

NCAM1 and NSE expression in the Beltran, et al. 2016 cohort (10), and (G) correlation with

773

CHGB and CHGA expression in the Kumar et al. 2015 cohort (25). See also Fig. S4.

774

Figure 6. HP1α represses AR and REST expression and enriches H3K9me3 on their 775

promoters. (A-B) AR and REST expression in stable V16D cells overexpressing HP1α as 776

determined by (A) qRT-PCR and (B) Western blotting. Bar graphs show mean ± SEM. The 777

p-values were calculated by unpaired two-tail Student’s t-test. (C) A heatmap comparing

778

expression of AR signaling genes in the indicated V16D cells. The select AR target genes 779

downregulated by HP1α overexpression are similarly downregulated in clinical NEPCs in the 780

Beltran, et al. 2011 cohort (23). (D) GSEA of V16D cells with stable HP1α overexpression. 781

Expression of pericentric heterochromatin components are upregulated by HP1α 782

overexpression. “NES” stands for normalized enrichment score; FDR q values were 783

calculated using 1000 gene permutations. (E) ChIP-PCR shows the enrichment of H3K9me3 784

on the promoters of AR and REST in V16D cells with HP1α overexpression, NC is a 785

negative control region. Bar graphs show mean ± SEM. The p-values were calculated by 786

unpaired two-tail Student’s t-test. (F) GSEA show the enrichment of pericentric 787

heterochromatin genes in human NEPC samples and mouse NE-like tumors. The y-axis 788

represents normalized enrichment score (NES) and the x-axis denotes clinical cohorts and 789

GEM models. FDR is less than 0.15 calculated by 1000 permutations. (G-H) Pearson 790

correlation analysis of HP1α mRNA expression and AR and REST mRNA levels from (G) 791

the Beltran et al. 2016 (10) and (H) the Kumar et al. 2016 cohorts (25). See also Fig. S5. 792

Published OnlineFirst February 27, 2018. Cancer Res

Xinpei Ci, Jun Hao, Xin Dong, et al.